Establishment and characterization of CRISPR/Cas9-mediated NF2(-/-) human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma
Establishment and characterization of CRISPR/Cas9-mediated NF2(-/-) human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma
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CRISPR/Cas9介导的NF2(-/-)人间皮细胞系的建立和表征：恶性胸膜间皮瘤中成纤维细胞生长因子受体2的分子洞察
DOI：
10.1111/cas.13871
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发表时间：
2019
期刊：
影响因子：
5.7
通讯作者：
Hosokawa Yoshitaka
中科院分区：
文献类型：
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作者：
Wahiduzzaman Md;Karnan Sivasundaram;Ota Akinobu;Hanamura Ichiro;Murakami Hideki;Inoko Akihito;Rahman Md Lutfur;Hyodo Toshinori;Konishi Hiroyuki;Tsuzuki Shinobu;Hosokawa Yoshitaka
恶性胸膜间皮瘤（Malignant pleural mesothelioma，MPM）是一种高度难治性肿瘤，由于缺乏早期诊断方法和药物治疗，目前无法治愈，这两种方法都是提高患者生存率和/或生活质量的迫切需要。使用CRISPR/Cas9系统，我们产生了NF 2敲除的人间皮细胞系MeT-5A（NF 2-KO）。在NF 2-KO细胞克隆中，与NF 2-WT细胞克隆相比，细胞生长、克隆形成活性、迁移活性和侵袭活性显著增加。互补DNA微阵列分析清楚地揭示了NF 2-WT和NF 2-KO细胞克隆之间的整体基因表达谱的差异。定量PCR分析和蛋白质印迹分析表明，成纤维细胞生长因子受体2（FGFR 2）的上调伴随着NF 2-KO细胞克隆中JNK、c-Jun和视网膜母细胞瘤（Rb）磷酸化水平的增加。这些增加都被NF 2-KO克隆中NF 2的外源性表达所消除。此外，NF 2-KO细胞克隆中FGFR 2的破坏抑制了细胞增殖以及JNK、c-Jun和Rb的磷酸化水平。值得注意的是，发现FGFR 2在NF 2阴性人间皮瘤组织中高度表达（11/12例，91.7%），但在NF 2阳性组织中表达较少。总之，这些发现提示NF 2缺乏可能通过介导FGFR 2的表达在人间皮肿瘤的发生中发挥作用; FGFR 2将成为开发针对NF 2缺失的MPM的治疗和诊断策略的候选分子。
Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients.NF2is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated anNF2‐knockout human mesothelial cell line, MeT‐5A (NF2‐KO). In NF2‐KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2‐WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2‐WT and NF2‐KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c‐Jun, and retinoblastoma (Rb) in NF2‐KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2‐KO clone. In addition, the disruption ofFGFR2in the NF2‐KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c‐Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2‐negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2‐positive tissues. Collectively, these findings suggest thatNF2deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM withNF2loss.